Process for preparing amino acids from hydrocarbons

ABSTRACT

A process for producing an amino acid, such as L-glutamic acid, L-lysine, L-ornithine, L-valine and L-homoserine, by fermentation from hydrocarbons as the main carbon source. The process is conducted by culturing a mixture of a microorganism capable of assimilating hydrocarbons and a microorganism capable of producing amino acids under aerobic conditions in an aqueous nutrient medium containing at least one hydrocarbon. Large yields of amino acids may be obtained by conducting a mixed culture of, for example, Micrococcus glutamicus with a hydrocarbonassimilating microorganism such as Arthrobacter paraffineus or Brevibacterium ketoglutamicum.

I United States Patent 1151 3,655,510 Tanaka et al. [451 Apr. 11, 1972[54] PROCESS FOR PREPARING AMINO [56] References Cited ACIDS FROMHYDROCARBONS UNITED STATES PATENTS [72] inventors: Katsunobu Tanaka;Kazuo Kimura, both fM lZu ae a ac I a s 3,389,058 6/1968 Kinoshita m1.......195/111 [73] Assignee: Kyowa Hakko Kogyo Co., Ltd., Tokyo, 3,411,990 11/1968 Udagawa et al. ..195/47 Japan Primary Examiner-A. LouisMonacell [22] June 1968 Assistant ExaminerGary M. Nath 21 1 App]. No:736,958 AttorneyCraig, Antonelli & Hill Related US. Application DataABSTRACT [63] Continuation of Ser. No. 695,329, Jan. 3, 1968, aban- Aprocessfor P u n an amino acid, such as -g clonal acld, L-lyslne,L-ormthine, L-valine and L-homoserme, by fermentation from hydrocarbonsas the main carbon source. The process is conducted by culturing amixture of a microorgan- [30] Fomgn Apphcauon Pnomy Dam ism capable ofassimilating hydrocarbons 1and a microorgan- Jan. 10, 1967 Japan ..42/1579 ism capable of producing amino acids under aerobic conditions in anaqueous nutrient medium containing at least one 52 us. c1. ..195/28 R195/1 11 hydrocarbon Large Yields Ofamino acids may be Obtained by [51]Int Cl 12b conducting a mixed culture of, for example, Micrococcus glu-[58] Field 4 47 111 tamicus with a hydrocarbon-assimilatingmicroorganism such as Arthrobacter paraffineus or Brevibacteriumketoglutamicum.

14 Claims, No Drawings PROCESS FOR PREPARING AMINO ACIDS FROMHYDROCARBONS CROSS-REFERENCE TO RELATED APPLICATION This application isa continuation of copending application Ser. No. 695,329, filed on Jan.3, 1968 now abandoned.

This invention relates to a process for producing amino acids. Moreparticularly, it relates to a process for the production of amino acidsby fermentation. Even more particularly, the invention relates to aprocess for the production of various amino acids by fermentation fromhydrocarbons as a raw material.

Recently, methods have been developed for ferrnentatively producingvarious useful substances from hydrocarbons using the so-calledhydrocarbon-assimilating microorganisms which are capable of growing andproliferating in hydrocarbons serving as the main source of carbon.However, the type of fermentation product which may be obtained therebyhas been limited up to now. Moreover, in most cases, a sufficientproduction yield has not been obtained. I

The present invention is directed to a process for producing aminoacids, which are widely used in many fields, using hydrocarbons as astarting material, hydrocarbons being relatively inexpensive.Accordingly, the present process has an applicability for large scaleproduction.

One of the objects of the present invention is to provide an improvedprocess for the preparation of amino acids which overcomes thedisadvantages and deficiencies of the prior art methods.

Another object of the present invention is to provide a process forproducing amino acids by fermentation may be carried out in anefficacious and simple manner.

A further object of the invention is to provide a method for thepreparation of various amino acids by fermentation which may be carriedout advantageously on an industrial scale using inexpensive startingmaterials to give a high yield of product.

These and other objects and advantages of the present invention willbecome apparent to those skilled in the art from a consideration of thefollowing specification and claims.

In accordance with the present invention, the present inventors havefound that various useful substances, particularly amino acids, can beproduced in high yields from a culture medium using at least-onehydrocarbon as the main source of carbon by culturinghydrocarbon-assimilating microorganisms together with microorganismswhich have no or only a weak hydrocarbon-assimilating ability. Byculturing together a microorganism capable of assimilating hydrocarbonsand a microorganism capable of producing amino acids, it is possible toprepare amino acids in the culture liquor in high yield.

A wide range of hydrocarbon-assimilating microorganisms can be utilizedin the process of the present invention since the direct producibilityof amino acids is not taken into account with respect thereto. Thus, asto the hydrocarbon-assimilating microorganisms used in the presentmethod, a wider range thereof can be employed than the case where aminoacids are produced by a single assimilating microorganism. This pointrepresents one of the greatest characteristics of the present invention.Another primary advantage of the present invention is the fact thatvarious amino acids can be produced by suffrciently utilizinghydrocarbons which have been heretofore deemed difficult or inadequateto use in the case of a fermentation process wherein a singleassimilating microorganism is employed. By the same token, unpurifiedraw materials may also be readily employed herein.

The scope of the present invention is not to be limited by the veracityof any theoretical explanation thereof, however, the present inventorshave hypothesized that the fermentation mechanism of the present methodis presumably based upon the following steps. The hydrocarbon isoxidized and decomposed to metabolic intermediates, for example, organicacids in the tricarboxylic acid cycle by the hydrocarbon-assimilatingmicroorganism and, then, the resulting intermediates are converted intoa useful amino acid by the action of the mixed microorganisms. It hasbeen further observed that, even when a hydrocarbon-assimilatingmicroorganism capable of producing an amino acid directly is used, theamino acid can be produced in a considerably higher yield in the casewhere a suitable amino acid-producing microorganism is selected andcultured together therewith, than in the case of a single culture. Thisfact is possibly ascribable to an efficient progress of metabolizationbrought about by a mutual supplementation of the metabolic intermediate,which often tends to become short.

Assimilating microorganisms belonging to genera such as Pseudomonas,Mycobacterium, Achromobacter, etc., in addition to thehydrocarbon-assimilating microorganisms which have been reportedheretofore, i.e., Corynebacterium, Arthrobacter, Brevibacterium,Micrococcus, etc., can be used in the present invention. Examples of theresults of fermentation carried out with combinations of thesehydrocarbon-assimilating microorganisms with various kinds of aminoacidproducing microorganisms are shown in Table 1.

These tests were carried out as follows. The hydrocarbonassimilatingmicroorganism was pre-propagated in 20 ml. of a culture medium preparedin a 250 ml. flask and containing, by

weight, 0.25% of yeast extract, 0.5% of meat extract, 0.5% of peptoneand 0.25% of sodium chloride, the medium having a pH of 7.2. Thepre-propagation was effected with aerobic shaking for 24 hours.

Likewise, the amino acid-producing microorganism was pre-propagated in20 ml. of a culture medium containing 2% of glucose, 1% of peptone, 0.5%of meat extract and 0.25 percent of sodium chloride, the medium having apH of 7.2.

200 ml. of a fermentation medium containing 10% of hydrocarbons amixture of n-paraffins having 9 to 18 carbon atoms), 0.1% of KH PO 0.1%of Na HPO '12l-l 0,0.05% of MgSO '7l-1 O, 0.002% of MnSo '4H O, 0.01% ofFeSO '71-l 0, 0.001% of ZnSo '7l-l O, 1% of (NI-10 0.1% of corn steepliquor, 1.0% of NZ-Amine (a trademark for a series of caseinhydrolysates) and 2.5 y/l of biotin was prepared in a 2 liter flask. Thetwo kinds of microorganism pre-propagated for 24 hours were inoculatedinto the fermentation medium at the same time, and culturing was carriedout at 30 C. with aerobic shaking for 4 days. The pH was adjusted tonear 7.2 with a urea solution during culturing.

In the examples as shown in Table 1, the two kinds of seed cultures weremixed in equal volumes and inoculated at the same time. It is to beunderstood, however, that the most suitable mixing ratio and the time atwhich either or both of the microorganisms is added to the fermentationmedium can be selected in each case, and the mixing ratio and time arenot restricted to any particular or specific limits.

Either a synthetic culture medium or a natural nutrient medium issuitable for carrying out the fermentation process of the presentinvention as long as it contains the essential nutrients for the growthof the particular strains employed. Such nutrients are well known in theart and include substances such as a carbon source, a nitrogen source,inorganic compounds and the like which are utilized by themicroorganisms employed in appropriate amounts. As a nitrogen source,various kinds of inorganic or organic salts or compounds, such as ureaor ammonia or ammonium salts such as ammonium sulfate, ammoniumchloride, ammonium nitrate, etc., or natural substances containingnitrogen such as corn steep liquor, peptone, meat extract, yeastextract, casein hydrolysates, fish meal, etc., may be employed. Mixturesof two or more of these substances may be used. Inorganic compoundswhich may be added to the culture medium include magnesium sulfate,potassium dihydrogen phosphate, potassium monohydrogen phosphate, sodiumchloride, iron sulfate, as well as other salts of magnesium, ironmanganese, zinc, calcium and the like. Mixtures of such inorganiccompounds may be employed. Also, nutrients essential for the growth ofthe particular strains employed are added to the medium, for example,vitamins such as biotin, thiamine, pantothenic acid, nicotinic acid,etc.

Hydrocarbons are employed in the process of the present invention as themain source of carbon. Hydrocarbons which may be used includestraightand branched-chain paraffins (alkanes) having from nine to 18carbon atoms, for example, n-nonane, n-decane, n-dodecane, n-hexadecane,n-octadecane, isodecane, etc. cycloparaffins, such as cyclohexane andcyclooctane, straightand branched-chain olefins such as pentene-2,hexene-l, octene-1octene-2, etc., cycloolefins such as cyclohexene,aromatic hydrocarbons such as benzene, o-xylene, p-xylene, etc., andmixtures thereof, and mixed hydrocarbons such as kerosene, light oils,heavy oils, paraffin oils, etc.

Small amounts of other carbon sources such as glucose, fructose,mannose, galactose, sucrose, starch, starch hydrolysate, waste molasses,etc. may be used in the fermentation medium along with the hydrocarbons.

The fermentation of the mixed cultures is conducted under aerobicconditions, such as aerobic shaking of the culture or with stirring of asubmerged culture, at a temperature of about 20 to 40 C. and a pH ofabout 4.0 to 9.0 After the completion of fermentation, the resultantamino acids are separated from the fermentation liquor by conventionalmeans, such as ion exchange resin treatment, extraction with solvents,precipitation with metallic salts, chromatography, or the like.

The following examples are given merely as illustrative of the presentinvention and are not to be considered as limiting. Unless otherwisenoted, the percentages therein and throughout the application are byweight per liter of water.

EXAMPLE 1 Arthrobacler paraffineus ATCC 15591 is inoculated into 20 ml.of a culture medium containing 0.25% of yeast extract, 0.5% of meatextract, 0.5% of pepton, and 0.25% of sodium chloride contained in a ml.flask. This medium has a pH of 7.2.

Micrococcus glutamicus 541 ATCC 13058 is inoculated into 20 ml. of aculture medium prepared in another 250 ml. flask and containing 2% ofglucose, 0.5% of meat extract, 1% of peptone and 0.25% of sodiumchloride. This nutrient medium also has a pH of7.2.

Both microorganisms are cultured at 30 C. with aerobic shaking for 24hours. The resultant cultures are then inoculated into 200 ml. of afermentation medium contained in a 2 liter flask and having thefollowing composition:

10% hydrocarbons (a mixture of n-paraffins of 9 to 18 carbon atoms) 0.1%KH PO,

0.1% Na HPO.,-12H 0 0.002% MnSO,*4H O 0.1% corn steep liquor This mediumhas a pH of 7.2.

Fermentation is then conducted by culturing the mixture ofmicroorganisms at 30 C. with aerobic shaking for 4 days. The decrease inpH is adjusted to close to 7.2 by adding Nl-LOH to the medium whileculturing. At the completion of fermentation, 35 mg./ml. of L-glutamicacid is formed in the culture liquor.

Calcium hydroxide is added to the fermentation liquor to adjust the PHthereof to 9.0, and then the medium is heated. After the microorganismcells have been filtered off, the filtrate is concentrated. By adjustingthe pH to 3.2 with hydrochloric acid and cooling the concentratedfiltrate, the calcium salt of L-glutamic acid is crystallized out. Byfiltering the resulting crystals, 5.2 grams of the calcium salt ofL-glutamic acid is obtained.

EXAMPLE 2 Brevibacterium ketoglutamicum ATCC 15588 is inoculated into 20ml. of a nutrient medium containing 0.25% of yeast extract, 0.5% of meatextract, 0.5% of peptone and 0.25% of sodium chloride, and having a pHof 7.2, which is prepared in a 250ml. flask. Micrococcus glutamicus 901ATCC 13287 is inoculated into 20 ml. of a medium containing 2% ofglucose, 1% of peptone, 0.5% of meat extract and 0.25% of sodiumchloride, and having a pH of 7.2, which is prepared in another 250 ml.flask.

Both microorganisms are cultured at 30 C. with aerobic shaking for 24hours. The resultant cultures are then inoculated into 200 ml. of afermentation medium contained in a 2 liter flask and comprising thefollowing components:

10% hydrocarbons (a mixture of n-paraffins of 9 to 18 carbon atoms) 0.1%KH PO 0.1% Na HPO 1 21-1 0 0.002% MnSO '4H O 0.01% FeSO.,'7H O 0.002%ZnSO '7H O 0.1 percent corn steep liquor 1 percent NZ-Amine 107/1 biotinThis medium has a pH of 7.2

Fermentation is then carried out with the mixture of microorganisms byculturing at 30 C. under aerobic shaking for 4 days. The decrease in thepH of the medium is adjusted to near 7.2 by adding ammonium hydroxide tothe medium while culturing. At the completion of fermentation, 2.0mg./ml. of L-lysine is formed in the culture medium.

After the microorganism cells have been filtered off by centrifugalseparation, the filtrate is acidified with hydrochloric acid. TheL-lysine is purified and separated in accordance with a conventionalmethod using an ion exchange resin. As a result, 0.31 grams of L-lysinehydrochloride is obtained.

EXAMPLE 3 Pseudomonas aeruginosa ATCC 15246 is inoculated into 20 ml. ofa nutrient medium containing 0.25percent of yeast extract, 0.5 percentof meat extract, 0.5 percent of peptone and 0.25 percent of sodiumchloride, and having a pH of 7.2, which was prepared in a 250 ml. flask.Culturing is conducted at 30 C. with aerobic shaking for 24 hours. Theresultant culture is then inoculated into 200 ml. of a fermentationmedium contained in a 2 liter flask and having the followingcomposition:

10 percent kerosene 0.1% KH PO 0.05% NgSO.,-7H O 0.002% MnSO -4H O 0. 1percent corn steep liquor This medium has a pH of 7.2.

Culturing is then conducted at 30 C. with aerobic shaking for 4 days.After 24 hours, a culture of Micrococcus glutamicus 541 ATCC 13,058 isadded to the fermentation medium. This latter culture is obtained byinoculating Microcaccus glutamicus 541 ATCC 13,058 into m1. of a mediumcontained in another 250 ml. flask and comprising 2percent of glucose, 1percent of peptone, 0.5 percent of meat extract and 0.25 percent ofsodium chloride, the medium having a pH of 7.2, and culturing at C. withaerobic shaking for 24 hours. During the fermentation of the mixedcultures, the lowering in the pH of the medium is adjusted to near 7.2by adding NH OH to the medium. At the completion of fermentation 12mg./ml. of L- glutamic acid is found to be formed in the fermentationliquor.

After filtering off the microorganism cells by centrifugal separation,the filtrate is concentrated. By adjusting the pH to 3.2 withhydrochloric acid, and cooling the filtrate, 2.1 grams of raw crystalsof L-glutamic acid is obtained. By recrystallization thereof, 1.5 gramsof L-glutamic acid is obtained.

It should be clear from the above that the principle of the presentinvention involves the preparation of amino acids by fermentation usinghydrocarbons as the main source of carbon, wherein a microorganismcapable of assimilating hydrocarbons and a microorganism capable ofproducing an amino acid are cultured together in mixture. Accordingly,to the present invention is not limited to any particular types ofmicroorganisms, but is applicable to all microorganisms which fallwithin these two defined categories. Microorganisms capable ofassimilating hydrocarbons are known in the art, and some of these havebeen exemplified above. Likewise, microorganisms capable of producingamino acids by fermentation are well known in the art, and these are allreadily employable in the process of the present invention.

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention, and all suchmodifications are intended to be included within the scope of thefollowing claims.

We claim:

1. A process for producing an amino acid by fermentation fromhydrocarbons which comprises culturing a mixture of 1) a microorganismcapable of assimilating hydrocarbons and (2) a microorganism capable ofproducing amino acids, microorganism (1) and microorganism (2) beingdifferent microorganisms, under aerobic conditions in an aqueousnutrient medium containing at least one hydrocarbon as the main carbonsource, accumulating the amino acid in the resultant culture liquor, andrecovering said amino acid from said culture liquor.

2. The process of claim 1, wherein the hydrocarbon-assimilatingmicroorganism belongs to a genus selected from the group consisting ofPseudomonas, Mycobacterium, Achromobacter, Corynebacterium,Arthrobacter, Brevibacterium and Micrococcus.

3. The process of claim 1, wherein the amino acid-producingmicroorganism is Micrococcus glutamicus.

4. The process of claim 3, wherein said amino acid is selected from thegroup consisting of L-glutamic acid, L- lysine, L-ornithine, L-valineand L-homoserine.

5. The process of claim 1, wherein the hydrocarbon used as the maincarbon source is an N-paraffin having from 9 to 18 carbon atoms.

6. The process of claim 1, wherein seed cultures of both microorganismsare added at the same time to said nutrient medium.

7 The process of claim 1, wherein seed cultures of the microorganismsemployed are added to the nutrient medium at different times.

8. The process of claim 1, wherein the amino acid-producingmicroorganism belongs to the genus Micrococcus.

9. A process for producing an amino acid by fermentation fromhydrocarbons which comprises culturing a mixture of 1) Micrococcusglutamicus and 2) a microorganism capable of assimilating hgdrocarbonsand belongin to a genus selected from te group consisting of seudomonas,

Mycobacterium, Achromobacter Corynebacterium, Arthrobacter,Brevibacterium and Micrococcus, microorganism (1) and microorganism (2)being different microorganisms, under aerobic conditions in an aqueousnutrient medium containing at least one hydrocarbon as the main carbonsource, accumulating the amino acid in the resultant culture liquor, andrecovering said amino acid from said culture liquor.

10. The process of group 9, wherein said amino acid is selected from thegroup consisting of L-glutamic acid, L- lysine, L-ornithine, L-valineand L-homoserine.

11. The process of claim 9, wherein the hydrocarbon used as the maincarbon source is an N-paraffin having from nine to 18 carbon atoms.

12. The process of claim 9, wherein culturing is carried out at atemperature of about 20 to 40 cC.nd at a pH of about 4.0 to 9.0.

13. A process for producing an amino acid by fermentation fromhydrocarbons which comprises culturing a mixture of (l) Micrococcusglutamicus ATCC 13,058 or Micrococcus glutamicus ATCC 13,287 and(2)Arthr0bacter paraflineus ATCC 15,591, Brevibacterium KetaglutamicumAtcc 15,588 or Pseudomonas aeruginosa ATCC 15,246 under aerobicconditions in an aqueous nutrient medium containing at least onehydrocarbon as the main carbon source at a temperature of about 20 to 40C. and at a pH of about 4.0 to 9.0, accumulating the amino acid in theresultant culture liquor, and recovering said amino acid from saidculture liquor. 1 8

14. The process of claim 13, wherein said amino acid is selected fromthe group consisting of L-glutamic acid, L- lysine, L-ornithine,L-valine and L-homoserine.

2. The process of claim 1, wherein the hydrocarbon-assimilatingmicroorganism belongs to a genus selected from the group consisting ofPseudomonas, Mycobacterium, Achromobacter, Corynebacterium,Arthrobacter, Brevibacterium and Micrococcus.
 3. The process of claim 1,wherein the amino acid-producing microorganism is Micrococcusglutamicus.
 4. The process of claim 3, wherein said amino acid isselected from the group consisting of L-glutamic acid, L-lysine,L-ornithine, L-valine and L-homoserine.
 5. The process of claim 1,wherein the hydrocarbon used as the main carbon source is an N-paraffinhaving from 9 to 18 carbon atoms.
 6. The process of claim 1, whereinseed cultures of both microorganisms are added at the same time to saidnutrient medium. 7 The process of claim 1, wherein seed cultures of themicroorganisms employed are added to the nutrient medium at differenttimes.
 8. The process of claim 1, wherein the amino acid-producingmicroorganism belongs to the genus Micrococcus.
 9. A process forproducing an amino acid by fermentation from hydrocarbons whichcomprises culturing a mixture of (1) Micrococcus glutamicus and (2) amicroorganism capable of assimilating hydrocarbons and belonging to agenus selected from the group consisting of Pseudomonas, Mycobacterium,Achromobacter Corynebacterium, Arthrobacter, Brevibacterium andMicrococcus, microorganism (1) and microorganism (2) being differentmicroorganisms, under aerobic conditions in an aqueous nutrient mediumcontaining at least one hydrocarbon as the main carbon source,accumulating the amino acid in the resultant culture liquor, andrecovering said amino acid from said culture liquor.
 10. The process ofgroup 9, wherein said amino acid is selected from the group consistingof L-glutamic acid, L-lysine, L-ornithine, L-valine and L-homoserine.11. The process of claim 9, wherein the hydrocarbon used as the maincarbon source is an N-paraffin having from nine to 18 carbon atoms. 12.The process of claim 9, wherein culturing is carried out at atemperature of about 20* to 40* cC.nd at a pH of about 4.0 to 9.0.
 13. Aprocess for producing an amino acid by Fermentation from hydrocarbonswhich comprises culturing a mixture of (1) Micrococcus glutamicus ATCC13,058 or Micrococcus glutamicus ATCC 13,287 and (2)Arthrobacterparaffineus ATCC 15,591, Brevibacterium Ketoglutamicum Atcc 15,588 orPseudomonas aeruginosa ATCC 15,246 under aerobic conditions in anaqueous nutrient medium containing at least one hydrocarbon as the maincarbon source at a temperature of about 20* to 40* C. and at a pH ofabout 4.0 to 9.0, accumulating the amino acid in the resultant cultureliquor, and recovering said amino acid from said culture liquor. 18 14.The process of claim 13, wherein said amino acid is selected from thegroup consisting of L-glutamic acid, L-lysine, L-ornithine, L-valine andL-homoserine.